Technology: Protein packs a punch in the fight against cancer

Genetically engineered drugs could soon be having a major impact – quite
literally – on cancer. The drugs, based on bacterial proteins that punch
holes in the blood cells of rabbits, can also puncture tumour cells. Once
perforated, the cells either die, or are left vulnerable to other, toxic
drugs which could be administered separately.

Hagan Bayley and his colleagues at the Worcester Foundation for Experimental
Biology in Shrewsbury, Massachusetts, have just begun adapting the bacterial
protein, called alpha-haemolysin, for use against human cancer cells. By
modifying the protein with genetic engineering, the investigators have already
found ways to switch its ability to perforate the cell walls on and off.

They want to modify the protein so that when it is injected into patients
it remains inactive until it meets a cancer cell. The idea is to adapt the
protein to be activated by enzymes called metalloproteinases that are produced
almost exclusively by tumour cells. ‘The molecule would be activated right
on the surface of the tumour cell,’ explains Bayley. ‘The pore-forming proteins
might kill the cells directly, although cancer cells may be able to repair
themselves. But you might be able to perforate the membrane and get drugs
inside that kill the cells.’ In theory, healthy cells without holes should
be unaffected by the toxic drugs.

Alpha-haemolysin is produced by the bacterium Staphylococcus aureus.
It punctures the blood cells of rabbits, but not of humans. Structurally,
the protein has two distinct domains (see Diagram). These are connected
by a bridging ‘loop’ which plays a critical role in the protein’s ability
to puncture a cell.

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Six molecules of the protein are needed to make each hole: they settle
on the cell’s surface, then link tightly together in a hexagonal shape.
After some time – still undetermined – the hole appears at the centre of
the hexagon. ‘We can see the assemblies with an electron microscope,’ says
Bayley. ‘They look like doughnuts.’

Bayley and his colleagues have not yet worked out how the molecules
join together, or how they punch the hole. But they have established that
the ‘loop’ region of the protein enables the pore to form, and then embeds
itself in the pore’s lining. By altering the loop via genetic engineering,
they have found ways to trigger pore formation, or to regulate what will
go through the pore. ‘We are trying to direct these proteins to specific
cells, and activate them when and where we like,’ says Bayley.

One modified version already works. The team anchored a short chain
of amino acids – a peptide – to the loop region. Bayley designed the peptide
so that it could be ‘clipped off’ by enzymes such as trypsin, a pancreatic
enzyme which is also made commercially for biological research. Once the
peptide is clipped off to expose the loop, the pore-forming activity of
the alpha-haemolysin is triggered.

Bayley showed that the system worked by mixing the genetically engineered
version in a test tube with red blood cells from a rabbit. Nothing happened
until the trypsin was added: then the blood cells began leaking profusely,
showing that the pore-forming activity of the modified protein had been
switched on.

Now Bayley and his colleagues are trying to work with metalloproteinases.
‘Metastatic tumour cells secrete these enzymes and they degrade tissue,
allowing the cells to burrow their way out of one organ and migrate through
the bloodstream to another,’ says Bayley.

The goal will be to design a peptide chain that is only attacked and
detached from the loop by metalloproteinases, so that the protein only forms
pores in tumour cells. Bayley hopes to have a system that works in a test
tube within a year, and expects to begin animal experiments soon after.